The present invention relates to the detection materials in small concentrations, especially the detection of pathogens. In particular it relates to the detection of proteins, DNA and RNA in serum.
Known manual pathogen detection methods in research and clinical laboratories tend to have low accuracy, low sensitivity to pathogens and are subject to human error, both in carrying out the methods and in interpreting the results. Other methods, e.g. culturing methods, are not suitable for many pathogens. For example, tuberculosis has a very slow growth rate, which makes detection not easy or even not possible.
One immunologic method, which identifies an organism with a known antiserum, is widely used for pathogen detection. The accuracy of the method is relatively high but the sensitivity is relatively low, e.g. it needs about one million antigen-antibody complexes to clearly indicate the results. There are a number of other disadvantages of this method, which are known.
In a standard enzyme ELISA method for immunoassay, a tray with a plurality of wells, e.g. 96 wells, containing appropriate antibodies, is used. One of the wells is used as a positive control (with a positive antigen), while the remaining wells are used for testing patient""s sera. After addition of the serum samples, the wells are washed and a second antibody, which carries an enzyme, is added to the wells. After washing again, a substrate is added. The substrate and enzyme react, with a colour reaction. The colour yield from the reaction is indicative of the presence of the pathogen. The method is rife with possibilities for error. Human error can lead to some wells being washed twice or not at all, having reagents added twice or not at all, or wells being inadvertently contaminated with extraneous materials. For example, overwashing tends to flush all the components and create a false negative result, while an incomplete wash will provide detection from non-binding materials and yield false positive results. The control well can give no assurance that the results from any other well is indicative of the presence or otherwise of the pathogen under investigation. Additionally, colour differences from well to well give additional uncertainties with respect to interpretation of the results.
Most of the previous tests are demanding of time, skill and concentration. So much so, that in many jurisdictions the number of tests that can be conducted by one technician is limited by regulation. This serves to raise the cost of testing, as it is so labour dependent.
For all the above reasons, and more, a new method of detecting pathogens is desirable, which is accurate, reproducible, and is sensitive to determining if there is an error in the method.
The present invention provides a method for detecting the presence of at least two predetermined known materials in a test sample, wherein at least one of the predetermined known materials is a control material, wherein the method comprises:
a) introducing the test sample, which contains at least one control material, into a test column which has a snare for each predetermined known material, each snare having a capture material specific to the associated predetermined known material, which will bind with the associated predetermined known material to form a bound material;
b) washing the test column to remove any materials which have not been bound to the capture materials; and
c) detecting the presence of bound materials on each of the snares.
In one embodiment, the predetermined known materials, which are not control materials, are pathogens.
In another embodiment, step c) comprises adding a label material for each of the bound materials to form labelled bound materials and then detecting the labelled bound materials.
In a further embodiment, the method is for detecting the presence of at least two predetermined known DNA materials, wherein the capture materials are single strand capture DNA materials and the test sample has been denatured so that any predetermined known DNA materials are in single strand form prior to addition to the test column, and detection of the bound materials is accomplished by adding a label material for each of the bound materials to form labelled bound materials and then detecting the labelled bound materials.
In another embodiment, the label material for the control DNA material and the non-control DNA material is selected such that the label material is a single label material.
Another aspect of the invention provides a method for detecting the presence of a pathogen, comprising the steps of:
i) adding a sample, which contains at least one control material, to a column which has at least one control snare and at least one test snare, each of the control snares having thereon a first capture antibody for binding to the control material, and each of the test snares having thereon a pathogen capture antibody for binding to the pathogen for which detection is being sought, so that the control material binds with the first capture antibody to form a bound control material, and any pathogen present binds with the pathogen capture antibody to form a bound pathogen;
ii) adding a wash solution to the column to remove any unbound control material and any unbound pathogens;
iii) adding sufficient primary antibodies to the column, to bind with the control material and any bound pathogens, said primary antibodies having labels thereon;
iv) adding a wash solution to the column to remove any unbound primary antibodies;
v) adding a substrate which reacts with the labels to give off a detectable signal; and
vi) detecting any detectable signals from the labelled and bound control material and from any labelled and bound pathogen.
In one embodiment, the substrate and label are selected to give off a chemiluminescent signal.
In another embodiment, the substrate and label are selected to give off a fluorescent signal.
A further aspect of the invention provides a method for detecting the presence of a DNA in a sample, comprising the steps of:
i) denaturing the predetermined known DNA materials;
ii) adding a sample, which contains at least one denatured control DNA segment, to a column which has at least one control snare and at least one test snare, one of the control snares having thereon a first control single strand capture DNA segment for binding to the denatured control DNA segment, and one of the test snares having thereon a test single strand capture DNA segment for detecting the denatured test DNA segment for which detection is being sought, so that the denatured control DNA segment binds with the first control single strand capture DNA segment to form a double strand control DNA segment, and any denatured test DNA present binds with the test single strand capture DNA segment to form a double strand test DNA segment;
iii) adding a wash solution to the column to remove any unbound DNA;
iv) adding S1 nuclease to the column to destroy any single strand DNA;
v) adding a wash solution and a denaturing solution to the column to reform the first control single strand capture DNA segment and the test single strand capture DNA segment;
vi) adding DNA probes to provide detectable labels for the first control single strand capture DNA segment and the test single strand capture DNA segment formed in step v);
vii) adding a wash solution to the column to remove any unbound DNA probe;
viii) adding a substrate which reacts with the labels to give off detectable signals; and
ix) detecting any signals from the control and test snares.
In yet another embodiment the method comprises:
i) preparing a positive control DNA material from a DNA material for which detection is sought, by a process selected from the group consisting of a) inserting a control DNA sequence into the DNA material at a predetermined scission point in the first DNA material and b) removing a small fragment of DNA from the first DNA material at a predetermined scission point;
ii) denaturing a test sample which contains at least the positive control DNA material;
iii) adding the sample, which contains denatured positive control DNA material, to a test column which has at least one control snare and at least one test snare, one of the control snares having thereon a first control single strand capture DNA segment for binding to a portion of the positive control DNA material, and one of the test snares having thereon a test single strand capture DNA segment for detecting denatured test DNA segment for which detection is being sought, so that the positive control DNA material binds with the first control single strand capture DNA segment to form a bound positive control DNA material which also has an unbound single strand segment, and any denatured test DNA present binds with the test single strand capture DNA segment to form a bound test DNA material which also has an unbound single strand segment;
iv) adding a wash solution to the column to remove any unbound DNA;
v) adding DNA probes to provide detectable labels for attachment to the unbound segment of the bound positive control DNA material and the unbound segment of the bound test DNA material formed in step iii);
vi) adding a wash solution to the column to remove any unbound DNA probe;
vii) adding a substrate which reacts with the labels to give off detectable signals; and
viii) detecting any signals from the control and test snares.
In yet another embodiment, the method is for detecting the presence of at least one predetermined known RNA material in a test sample, wherein the test sample contains at least one predetermined known control DNA material, and wherein the test sample has been denatured so that any RNA and DNA materials are in single strand, wherein the method comprises:
In one embodiment, in the RNA method a second control DNA material is also present, in which the second control DNA material has a partial DNA sequence match with the associated capture DNA material, so that in step a) the second control DNA material forms a partially bound DNA material with single strands attached thereto, and so that in step c) the single strands attached to the partially bound material are destroyed, leaving partially bound DNA material, and so that in step d) a partial DNA sequence single strand material is formed by denaturing.
The present invention also provides a column for analysis of at least one pathogen in which the column has at least two snares, one of said snares having thereon a first control capture material for detecting the presence of a first control material, and the other of said snares having thereon a pathogen capture material for detecting a pathogen for which detection is being sought.
In one embodiment, the snares are separated longitudinally along the column.
In another embodiment, the snares are separated radially about a longitudinal axis of the column.
In yet another embodiment, column has a snare having thereon a first control material, and a plurality of snares each having thereon a pathogen capture material wherein the pathogen capture materials are different from one another.
In a further embodiment, the column has a snare-having thereon a first control material, at least one snare having thereon a pathogen capture material and a snare having a second control material for detecting the presence of a second control material.
In another embodiment, the capture materials are capture antibodies.
In a further embodiment, the capture materials are single strand DNA materials.
In yet another embodiment the presence of the first control material and the pathogen material may be detected by the same detection material.
In another embodiment, the column has a longitudinal axis and comprises at least two chambers, at least one of the chambers containing a snare with a control capture material, and at least one of the chambers containing a pathogen capture material, each of said chambers having connecting means to connect the chambers in a predetermined sequence along the axis.
In yet another embodiment, each chamber has a different cross-sectional areas of all other chambers.
In a further embodiment, each chamber has a different diameter from the diameters of all other chambers.
In yet another embodiment, the column has an additional chamber with a snare without a capture material thereon.
In another embodiment, the column has a longitudinal axis and comprises at least one chamber, each chamber containing at least one snare with a control capture material and at least one pathogen snare each with a pathogen capture material, wherein the snares are spaced radially about the longitudinal axis.
In another embodiment, the first control material is albumin.
In another aspect of the invention, there is provided a kit which comprises i) a column for analysis of at least one pathogen in which the column has at least two snares, one of said snares having thereon a first control capture material for detecting the presence of a first control material, and the other of said snares having thereon a pathogen capture material for detecting a pathogen for which detection is being sought, ii) reagents for detecting the presence of the materials selected from the group consisting of a) reagents for detecting the presence of the control pathogen and the test pathogen, and b) reagents for detecting the presence of the first control capture material and the pathogen capture material after the first control capture material and the pathogen capture material have been bound and then unbound from the first control material and the pathogen material.
In one embodiment, the kit also includes wash solutions for removing excess reagents from the column.
Another aspect of the invention provides an apparatus for detection of at least two predetermined known materials in a sample, at least one of said predetermined known materials being a control material, said apparatus comprising:
a) a sample addition station for adding a sample to a testing column;
b) at least one reagent addition station, each reagent addition station being associated with a washing station;
c) a detection station for detection of the predetermined known materials which may be in the testing column;
d) conveying means for conveying the testing column from the sample addition station, sequentially past each reagent addition station and washing station and thence to the detection station.
In one embodiment, each reagent addition station has delivery means for delivering a measured quantity of reagent to the column and each washing station has delivery means for delivering, a measured quantity of washing solution to the column.
In another embodiment, the column has a longitudinal axis and a plurality of snares, each for capturing a predetermined known material, and the detection station has a plurality of detectors for detecting the presence of the predetermined materials at the snare locations along the longitudinal axis.
In a further embodiment, the detection station has two detectors for detecting different control materials, and a sufficient number of detectors for detecting the predetermined known materials.
In another embodiment, the detectors are laser detectors for detecting chemiluminescence.
In yet another embodiment, the column has a plurality of snare locations in a plane transverse to a longitudinal axis of the column, each snare having means for capturing a predetermined known material, and the detection station has a plurality of detectors for detecting the presence of the predetermined materials at the snare locations.
In a further embodiment, the detectors are for predetermined known materials selected from the group consisting of pathogens, DNA materials and RNA materials.
Another aspect of the invention provides an apparatus for recycling used test columns which have at least one snare with a capture material attached to the snare and a test material attached to the capture material, comprising
a) a stripping station for adding a stripping material to the test column, in order to strip the test material from the capture material;
b) a first washing station after each stripping station, for adding a wash material in order to wash stripped test material from the column;
c) a first detection station for detecting the presence of the test material; and
d) conveying means for conveying test columns sequentially past each of the stripping station, washing station and detection station.
In one embodiment there is a second stripping station, second washing station and second detection station and the conveying means is for conveying the test column sequentially past each of the second stripping station, second washing station and second detection station after being conveyed past the first detection station.